HIV-1 particle assembly occurs on the plasma membrane of infected cells. The assembly process is directed by the Gag polyprotein, which is synthesized on cytoplasmic ribosomes, and travels to the plasma membrane assembly site by an incompletely understood pathway. Env proteins are synthesized on ribosomes of the rough endoplasmic reticulum, form trimers in the ER, and transit the secretory pathway of the cell to the plasma membrane. Despite following very different pathways, Gag and Env meet at the particle budding site, and Env is specifically concentrated at this site. New evidence presented here indicates that Env reaches the particle assembly site in a directed manner through interactions with a trafficking complex directed by Rab11- FIP1C (FIP1C) and Rab14. This project will define the mechanisms by which FIP1C and Rab14 direct Env sorting and incorporation onto HIV-1 particles. We will then dissect specific regulators of actin polymerization discovered in a unique siRNA screen that strongly affect budding or release of HIV-1 particles. Experiments in Aim 1 will first define FIP1C functional domains that specifically are required to translocate Env onto the particle, and then determine the motif(s) within the Env cytoplasmic tail involved in FIP1C-dependent trafficking and incorporation. TIRF microscopy and live cell imaging will be pursued to fully define the steps in FIP1C-Env trafficking to the particle assembly site. In Aim 2, the contribution of the FIP1C-Rab14 trafficking pathway to polarization of HIV structural components and to cell-cell transmission of virus will be addressed. Experiments in this aim will also identify the kinesin responsible for movement of the Env-FIP1C-Rab14 complex to the particle assembly site. We will then follow up on exciting new findings indicating that FIP1C mediates MA-Env interactions, testing two relevant models for Gag trafficking and Gag-Env interaction. Aim 3 will examine a related area, the contribution of ROCK1-LIMK1-cofilin pathway to particle budding and release, and will define the role of related actin regulatory proteins that modulate HIV particle production. This experimental plan will add significantly to the knowledge of specific host factors involved in HIV particle assembly and budding, and will provide fundamental new insights into cellular trafficking pathways utilized by enveloped viruses.